Polyketides are complex natural products that are abundant in soil bacteria. From fewer than 10,000 known polyketides, a large number of therapeutic agents have been derived, covering every important therapeutic area. Complex polyketides are synthesized by polyketide synthase (PKS) consisting of sets (modules) of catalytic domains acting sequentially to build long carbon chains. Rudimentary knowledge of the rules governing the ability of PKS modules or domains to interact productively exists but the level of understanding has negated many attempts to engineer desired polyketides. The long-term aim of this proposal is to overcome this problem and to determining how to construct almost any PKS, and thus any polyketide, by combining heterologous modules in a productive fashion. Its novelty lies in the analysis of hundreds or thousands of module combinations in a semi-empirical approach and selecting those that work well. This is enabled by our recent development of high-throughput long gene synthesis technology, and a design of generic PKS modules that each contains the same restriction sites at module and domain boundaries, enabling a "building block" approach to combinatorial biosynthesis. The knowledge base for rational PKS engineering will bring enormous benefits to the manufacture of desired therapeutics as well as the availability of fermentation-derived structures to be used as intermediates in chemical synthesis and manufacturing. The specific aims of this Phase I proposal are (1) to test functionality of 2-module hybrid PKS systems with >200 combinations and identify those that produce polyketide product and (2) to test the connectivity of the results obtained with overlapping modules in the biomodular system to rationally create larger polyketide chains. The resulting technology will be expanded and perfected in Phase II research for the production of natural products derived from plant, animal, and microbial sources, such as antibiotics, anticancer drugs, and other therapeutic agents, and useful synthetic starting materials for manufacturing polyketides of current interest.